Telomeric protein function and regulation Project Summary/Abstract Continued cell division mandates the maintenance of chromosome ends, which undergo shortening due to the inability of the replicative DNA polymerases to completely synthesize them. Telomerase is a unique ribonuclear protein enzyme that can bind chromosomes ends to extend them with repetitive sequences called telomeric DNA. Using this mechanism, telomerase can help reduce the erosion of chromosome ends and sustain the continued proliferation of actively dividing cells such as somatic stem cells, and cells that make up ~90% of human cancers. Whereas aberrant activation of telomerase in non-dividing somatic cells predisposes them to cancer, mutations in genes that reduce telomerase function in stem cells result in one of many premature aging diseases, including dyskeratosis congenita. Therefore understanding how telomerase function is tightly regulated in cells bears major implications for the biology of aging, and for diseases such as cancer. Telomeric DNA in the cells is not exposed, because this would result in its recognition and repair by the DNA damage response/repair machineries in the cell, leading to illicit end-to-end DNA-joining events at chromosome ends. Our cells therefore have in place a six-protein complex named shelterin that specifically binds telomeric DNA to protect it from illicit DNA repair events. TPP1 is a unique shelterin protein that is central to both telomerase function and chromosome end protection. However, how a single TPP1 gene facilitates the solutions of such distinct biological problems remains unknown. Using a multi-disciplinary approach that includes biochemistry, cell biology, single-molecule microscopy, X-ray crystallography, and bioinformatics, this proposal aims to understand how TPP1 upholds chromosome end protection and end replication. Aim 1 of the proposal will reveal at a molecular level how TPP1 helps protect the single-stranded regions of telomeric DNA with the help of the POT1 protein. Aim 2 of this proposal will involve a novel selective-knockout strategy using CRISPR- Cas9 technology, to ask how natural isoforms of TPP1 facilitate different aspects of end protection and end replication. Aim 3 will explore the mechanism-of-action and pervasiveness of novel RNAs that regulate TPP1 expression in human cells using RNA biochemistry, cell biology and bioinformatics approaches. These studies will reveal the mechanistic basis for how a single TPP1 gene can orchestrate both chromosome end protection and end replication, and discover previously unanticipated mechanisms by which noncoding RNAs regulate TPP1 abundance and function in human cells.